According to the catecholaldehyde hypothesis, cytoplasmic dopamine is converted by mitochondrial monoamine oxidase to the toxic catecholaldehyde, dihydroxyphenylacetaldehyde (DOPAL). DOPAL is detoxified by aldehyde dehydrogenase (ALDH), to form dihydroxyphenylacetic acid (DOPAC). Using a sensitive assay method for measuring DOPAL, DOPAC, and dopamine simultaneously, we found that in the putamen of patients with end-stage PD the amount of DOPAL per dopamine terminal is increased, and ALDH activity measured by the DOPAC:DOPAL ratio is decreased. These findings are consistent with DOPAL buildup and decreased ALDH activity in the putamen of PD patients. By applying a combined neuroimaging-neurochemical approach we recently obtained evidence for increased turnover and decreased vesicular sequestration of catecholamines in Lewy body diseases. We followed interventricular septal 6-18Ffluorodopamine-derived radioactivity over time in PD patients with or without orthostatic hypotension (OH), pure autonomic failure (PAF), multiple system atrophy (MSA), and normal control subjects with or without pre-treatment by desipramine to inhibit neuronal uptake, mimicking denervation. Arterial plasma levels of 6-18Ffluorodihydroxyphenylacetic acid (6-18FFDOPAC), the main neuronal metabolite of 6-18Ffluorodopamine, were measured in subgroups of PD+OH, PAF, MSA, and control subjects. We found that in the 8-25 minute interval, cardiac 6-18Ffluorodopamine-derived radioactivity declined mono-exponentially. Compared to MSA patients and normal controls, PD patients had lower y-intercepts (y0) and greater slopes of decline than did both comparison groups. Across groups, mean slopes varied inversely with y0. 6-18FFDOPAC:y0, an index of intraneuronal metabolism adjusted for neuronal uptake, was increased in PD+OH and PAF compared to MSA and desipramine-treated and untreated normal groups. From these results we conclude that Lewy body diseases feature accelerated loss of sympathoneural catecholamines, which denervation alone does not explain. High 6-18FFDOPAC:y0 ratios suggest pathophysiologically significant decreased vesicular sequestration of cytosolic catecholamines in these diseases. A manuscript describing these results is in preparation. We are making progress in developing a high sensitivity, rapid method for measuring levels of catecholamines using liquid chromatography with mass spectroscopy. We have successfully detected as low as 2 pg of native and deuterated norepinephrine, epinephrine, and dopamine simultaneously in 3-minute chromatographs and measured all three catecholamines in alumina extracts of plasma from supine, resting humans. In a collaborative study with Dr. Huaibin Cai (NIA) we have been assaying midbrain and striatal concentrations of dopamine and other catechols in mice with inherited expression of mutated alpha-synuclein or LRRK2 or with knockout of ALDH1A1 in various combinations. So far these models have not shown substantial decreases in striatal dopamine content. We initiated a collaboration with Randy Strong (Univ. of Texas) about brain and cardiac tissue concentrations of catechols in animals with double knockout of the genes encoding ALDH1A and ALDH2 and a collaboration with Gary Miller (Emory) about tissue catechols in animals with extremely low activity of the type 2 vesicular monoamine transporter. Both animal models are expected to increase cytosolic catecholamines and catecholaldehydes and produce motor and non-motor manifestations resembling those in PD. Preliminarily, mice with double knockout of ALDH1A and ALDH2 have neurochemical evidence for low striatal ALDH activity. In vitro studies have shown that DOPAL potently oligomerizes alpha-synuclein. We have recently found that metal ions (especially ionized copper) augment DOPAL-induced oligomerization of alpha-synuclein. These preliminary findings suggest that deleterious interactions of catecholaldehydes, metal ions, and alpha-synuclein might explain relatively selective loss of catecholaminergic neurons in Lewy body diseases. Our cellular studies have shown that reserpine, which blocks the vesicular monoamine transporter, builds up cytosolic endogenous dopamine, DOPAL, and other deaminated catecholamine metabolites in catecholamine-producing cells. Studies are under way about whether reserpine augments dopamine-induced apoptosis.